Our research program is focused on the analysis of the three-dimensional structures of macromolecular assemblies using high resolution electron microscopy. Members of our group work on different, yet, complementary aspects of structural analysis. These include electron crystallographic studies of two-dimensional protein crystals, "single particle" approaches to analyze the structures of protein complexes and determination of the structures of large subcellular assemblies using electron tomography. A significant fraction of our research effort is devoted to developing and implementing novel methods for specimen preparation, high throughput data acquisition and computational analysis. Research Highlights 2000-2001: ? We completed a comprehensive analysis of structural changes by bacteriorhodopsin that allowed us to present a detailed atomic mechanism for the light-driven transport of protons by this protein. Aside from the new biological insights, our atomic resolution structure for the open conformation of the protein is at the highest resolution (~ 3?) that has been obtained on any membrane protein by electron microscopy. ? We have successfully crystallized a bacterial oxalate transporter, and have determined a projection structure at 6 ? resolution. This work provides the first insights into the architecture of a very large superfamily of transporter proteins. ? We have developed methods to introduce a high level of automation in biological electron microscopy. This work has already had a considerable impact on the speed of data collection in our laboratory. NCI has filed a provisional patent application on this technology. ? We have derived an atomic model for the 11,000 kD pyruvate dehydrogenase complex from B. stearothermophilus. The model was obtained by docking in four individual components of the enzyme whose structures have been determined by X-ray crystallography into a medium resolution electron microscopic model of the entire complex. The results have been very exciting and unexpected. ? We have demonstrated that CCD detectors can now fully replace photographic film to record electron diffraction patterns to resolution of 2 ?, and have an active interest in continuing to improve detector technology for high resolution electron microscopes by testing bigger and better CCD cameras for molecular imaging applications. In the coming years, our plan is to continue to develop an infrastructure for high resolution electron microscopy at NCI/NIH and to address fundamental biological problems using three principal types of methodologies: ? Electron crystallography of membrane proteins, at near-atomic resolution with a particular focus on proteins that function as transporters in biological membranes ? Molecular electron microscopy to determine the architectures of large macromolecular complexes such as multi-protein cellular machines and complex assemblies with sizes ranging from 200 kD to 10, 000 kD by analysis of "single particle" images ? Electron tomography to determine the three-dimensional architectures of very large molecular assemblies, subcellular organelles and cellular sections Our laboratory is currently equipped with a 300 kV field emission gun, and two standard 120 kV electron microscopes. We also have a cluster of high end computing machines that include several DEC Alphas, SGIs, and Linux machines for image processing.